This invention relates to an apparatus and method and to system elements for forming capillary columns containing solid separation media within microfluidic circuits for effecting sample analysis, such as chromatography. More particularly, this invention relates to an apparatus and method for forming arrays of capillary channels in a microfluidic circuit, and devices for inserting and retaining solid separation media in the channels, and fluid inlet and fluid outlet connectors for interfacing with the capillary channels, and detection apparatus for monitoring elution gradients or analytes passing through the capillary channels.
Reducing the complexity of biological samples is crucial to the effectiveness of proteomic analysis. Biological samples contain many thousands of components, and a series of sequential separation steps are needed to isolate individual components for analysis. In this procedure, each fraction generated in a previous separation stage undergoes further fractionation. For example, if after the first separation stage 30 fractions are produced, then each of these fractions undergoes a full separation process in the second stage. Each one of the second-stage separations may produce between 30 and 200 fractions (for a total of between 900 and 6000 fractions). The number of sample fractions increases geometrically, but the number of separation processes also increases geometrically. This explosive increase in the amount of separation work is impractical when using current technology. In addition, dividing a single sample into large numbers of fractions can result either in unacceptable dilution of the fractions, or else in the necessity of handling ever-smaller volumes. As the sample volumes become extremely small, the prospect of serious sample losses becomes quite significant.
Conventional chromatography is generally carried out by the passage of a sample mixture through a bed of spherical beads. The beads are tightly packed in a tube or column in a manner to minimize interstitial volume between the beads so as to increase the separation efficiency of the column. The chromatography bed is designed to retard the passage of different components of the mixture to different degrees, resulting in separation of those components as the mixture proceeds through the chromatography bed. An alternative separation medium to chromatography beads is monolithic media which are monolithic blocks taking the form of the containment volume.
Chromatography is highly regarded as a method for separating complex mixtures because several different separation mechanisms are available (such as ion-exchange, reversed-phase, or affinity chromatography). The separation efficiency of well-designed chromatography methods can be quite high, and chromatography can be scaled over a very wide range of volumes. Chromatography would be the method of choice for reducing the complexity of very complex mixtures; however, chromatography lacks sufficient throughput, thus high throughput requires highly parallel operation.
Chromatography at its smallest scale is called capillary chromatography. This is usually conducted in discreet capillaries with internal diameters between 0.01 mm and 0.25 mm filled with packed beds of very small particles or beads. The complementary components required for capillary chromatography, such as column frits, flow cells, and sample injection valves, are also discreet devices to which the capillaries are attached. One of the limitations of capillary chromatography is that the complementary components are generally too large for the scale of the capillary bed, resulting in dispersion of the sample and blending of the separated peaks. Furthermore, the use of discreet components is impractical for arrays of capillary liquid chromatography (LC) systems because of the high cost and the large size of components.
Formation of capillary channels in microfluidic circuits is an attractive alternative to the use of discreet components for making capillary LC systems. As used herein microfluidic circuits refer to, fluid transport and control structures formed within a single substrate made from glass (preferably fused silica), ceramic or plastic materials. Microfluidic circuits enable all of the associated components of a chromatography system to be integrated into one device, appropriately scaled to minimize sample dispersion. Certain critical components required to assemble a chromatography system on a single microfluidic circuit substrate, or an array of systems on a single substrate, are not currently available or, if available, are commercially impractical. For example, the structure for retaining small particles of the chromatography separation medium in larger, conventional chromatography columns is a filter device (sometimes known as a filter frit) that is installed in the fluid path to hold back particles larger than some selected threshold. However, frits are not a viable solution for microfluidic circuit technology. It has been proposed to use devices called Weir filters in photolithographic approaches, but such devices are difficult to manufacture as the depth of the barrier is excessively sensitive to etching depth and this sensitivity has been an impediment to the Weir filter""s viability in microfluidic circuits.
An alternative to using small particles as a capillary chromatography medium is to utilize monolithic chromatography beds polymerized directly in place in a capillary. These monolithic media, composed of polymers such as styrene-divinylbenzene copolymers, are porous, uniform blocks taking the form of the containment volume. Examples of the use of such media for capillary chromatography applications are described in U.S. Pat. Nos. 5,130,343; 5,334,310 and 5,453,185. Pumping a mobile phase through the monolithic bed provides unusually fast chromatographic separations because the diffusion distance to the stationary phase is negligible (i.e., tens of nanometers). Just as with discrete separation particles, it is difficult to retain the precursor solution used to form the monolithic bed within the desired separation zone. The fluid precursor to the polymerized bed will tend to flow, pulled by capillary forces, throughout the capillary network rather than being confined to the defined separation zone. This result is undesirable since it is necessary to separate the fluid sample from the solid separation medium so that the fluid sample can be analyzed.
In addition to the problems associated with capillary chromatography set forth above, carrying liquids to a microfluidic circuit or from the microfluidic circuit to a destination off the circuit is difficult because the liquid volumes are extremely small. The transport tubing must be a discrete capillary with a small bore (e.g., 25-150 microns), and the capillary tubing must align and seal with a similarly small hole on the surface of the circuit. The alignment of the capillary to the hole on the circuit is a problem due to the small dimensions, and formation of a reasonably leak-tight seal under the high pressures chromatography systems typically operate is even more difficult. Moreover, at these small dimensions, the surface finish is often coarse compared to the features themselves and this adds to the difficulty in achieving an appropriate seal. A further complication is that the connections should preferably be reversible because large numbers of connections are anticipated and repair or replacement of defective capillaries is desirable. The method of coupling also should have a small footprint to permit high density coupling of connective tubing to the circuit. The coupling technique chosen also must be manufacturable, i.e., it must be possible to automate the process, or at least to assemble such fittings with a minimum of time and rework.
Accordingly, it would be desirable to provide a capillary chromatography process and system contained within a microfluidic circuit as well as system elements that solve the problems set forth above.
The present invention provides a capillary chromatography process and system contained within a microfluidic circuit that utilizes a solid separation medium confined to a desired capillary containment volume. In one aspect, the solid medium can be either discrete particles or a porous monolithic solid. The solid medium is retained within the desired containment volume by forming capillary paths within a plurality (i.e., 3 or more) of layers that make up the microfluidic circuit. The retainment structures are positioned adjacent to the containment volume of the separation capillary and are formed with channel depths that differ between adjacent layers, thereby creating dimensional restrictions to prevent passage of the discrete solid medium, or of a fluid precursor used to form the monolithic solid medium, but which permit the passage of a fluid sample to be separated by the solid medium.
In one embodiment, the chromatography system includes an array of capillary chromatography columns within the circuit. The use of photolithographic etching processes on fused silica substrates assures that each capillary chromatography element will be a replicate of all other such elements. As a result of using such techniques, a plurality (e.g., 16 or more) of samples can undergo chromatographic analysis in parallel in a highly repeatable fashion.
In still another aspect of the present invention, system connecting elements between discrete capillary transport tubes and the microfluidic circuit are used that allow introduction and removal of fluid streams from the array of capillary chromatographic columns while preventing dispersion of the fluid sample. The connections afford both precision alignment and high pressure alignment necessary to perform chromatography.